Method of treating endothelial injury

ABSTRACT

The use of human erythropoietin (EPO) to prevent or treat endothelial injury due to chemotherapy, radiation therapy, mechanical trauma, or to a disease state which damages the endothelium (such as inflammation, heart disease or cancer) is described. The use of EPO in conjunction with the administration of chemotherapeutic agents is described.

FIELD OF THE INVENTION

The present invention relates to the use of human erythropoietin (EPO)in the prevention or treatment of endothelial injury due tochemotherapy, radiation therapy, mechanical trauma, or to a diseasestate which damages the endothelium (such as inflammation, heart diseaseor cancer). The present invention further relates to the use of EPO inconjunction with chemotherapy.

BACKGROUND OF THE INVENTION

Erythropoietin (EPO) is a glycoprotein produced in the kidney, and isthe principal hormone responsible for stimulating red blood cellproduction (erythrogenesis). EPO stimulates the division anddifferentiation of committed erythroid progenitors in the bone marrow.Normal plasma erythropoietin levels range from 0.01 to 0.03 Units/mL,and can increase up to 100 to 1,000-fold during hypoxia or anemia.Graber and Krantz, Ann. Rev. Med. 29:51 (1978); Eschbach and Adamson,Kidney Intl. 28:1 (1985). Recombinant human erythropoietin (rHuEpo orepoetin alfa) is commercially available as Epogen® (Amgen Inc., ThousandOaks, Calif.) and as Procrit® (Ortho Biotech Inc., Raritan, N.J.). EPOis indicated for treatment of anemia, including anemias associated withcancer chemotherapy, chronic renal failure, malignancies, adult andjuvenile rheumatoid arthritis, disorders of haemoglobin synthesis,prematurity, and zidovudine treatment of HIV infection.

The vascular endothelium is a layer of cells lining the inner vascularwall and in direct contact with blood, providing an active naturalbarrier between the circulatory and extravascular compartment. Theendothelium is involved in signal and information transfer at thecellular, tissue and organ level, and plays a role in both cell-mediatedand humoral immune responses. Endothelial cells are metabolically activeand normally produce a number of substances with effects on the vascularlumen and on platelets. Endothelial vasodilators include prostacyclin(PGI₂) and endothelium-derived relaxing factor (EDRF, which may benitric oxide or a more stable adduct thereof); these two substances alsoact to inhibit platelet aggregation.

Damage or destruction of the endothelium by physical trauma or diseaseprocesses such as atherosclerotic plaque formation may impair EDRFproduction, contributing to vasoconstriction. More diffuse and subtleendothelial damage, such as due to chronic hypertension or reperfusionafter ischemia, also leads to altered EDRF production. Endothelialproducts localized to the luminal endothelial surface include ectoADPaseand thrombomodulin. Vasoconstrictors released by the endothelium includeendothelin. Endothelial cells also secrete growth factors which enhanceendothelial mitogenesis and can induce new blood vessel formation(angiogenesis). It has been reported that granulocyte macrophage-colonystimulating factor (GM-CSF) and granulocyte-colony stimulating factor(G-CSF) stimulate proliferation and migration of endothelial cells.Interleukin-3 (IL-3) also enhances the proliferation of these cells. SeeBussolino et al., Nature 337:471 (1989); Brizzi et al., J. Clin. Invest.91:2887 (1993).

SUMMARY OF THE INVENTION

A first aspect of the present invention is a method of reducingendothelial injury caused by a chemotherapeutic agent, by administeringan endothelial-protecting amount of erythropoietin in conjunction withthe administration of the chemotherapeutic agent. Theendothelial-protecting amount of erythropoietin may be administeredsimultaneously with the chemotherapeutic agent, prior to thechemotherapeutic agent, or after the chemotherapeutic agent.

A second aspect of the present invention is a method of enhancingendothelial cell inhibition in a subject treated with a chemotherapeuticagent, by administering an endothelial-inhibiting amount oferythropoietin in conjunction with the chemotherapeutic agent. Theendothelial-inhibiting amount of erythropoietin may be administeredsimultaneously with, prior to, or after the chemotherapeutic agent.

A further aspect of the present invention is a method of treating asolid vascularized tumor by administering an antineoplasticchemotherapeutic agent in conjunction with an endothelial-inhibitingamount of erythropoietin. The endothelial-inhibiting amount oferythropoietin may be administered simultaneously with, prior to, orafter the chemotherapeutic agent.

A further aspect of the present invention is a method of treatingendothelial injury caused by mechanical damage, exposure to radiation,inflammation, heart disease or cancer by administering anendothelial-protecting amount of erythropoietin to a subject in need ofsuch treatment.

The foregoing and other objects and aspects of the present invention areexplained in detail in the specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the dose-response curve for viability ofendothelial cells after exposure to cisplatin.

FIG. 2 is a graph showing the responses of endothelial cell culturesexposed simultaneously to cisplatin and varying dosages of EPO, comparedto control endothelial cell cultures exposed only to cisplatin.

FIG. 3 is a graph showing the responses of endothelial cell culturesexposed first to cisplatin and, two hours later, to varying dosages ofEPO (compared to control endothelial cell cultures exposed only tocisplatin).

FIG. 4 is a graph showing the responses of endothelial cell culturesexposed first to varying dosages of EPO and, two hours later, tocisplatin (compared to control endothelial cell culture exposed only tocisplatin).

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have previously shown that recombinant humanerythropoietin (EPO) exerts a mitogenic and chemoattractant (migratory)effect on human umbilical vein endothelial cells and bovine capillaryendothelial cells. Anagnostou et al., Proc. Natl. Acad. Sci. USA 87:5978(1990). Endothelial cell migration and proliferation are the key stepsin the angiogenic process.

The present inventors have found that EPO can effectively prevent and/orrepair endothelial damage caused by chemotherapeutic agents. The presentinventors have found that administration of EPO concomitantly withchemotherapeutic agents produces a biphasic response: certain doses ofEPO protect endothelial cells from the deleterious effects of thechemotherapeutic agent, while increased doses enhance the endothelialgrowth-suppression caused by the chemotherapeutic agent.

The use of EPO to enhance endothelial growth-suppression duringchemotherapy is useful in treating angiogenic tumors, where it isdesirable to prevent or slow the formation of new blood vessels whichsupport tumor growth. Tumors require an adequate blood supply, andgrowth of new vessels in the tumor mass is stimulated by angiogenicfactors secreted by tumor tissue. In animal models, inhibition ofangiogenesis in tumor tissue has been shown to cause tumor regression.Highly vascularized solid tumors include cerebellar hemangioblastoma,ductal carcinoma of the breast, and squamous cell cancer of the larynx.Abnormal angiogenesis is involved in additional pathological conditions,including diabetic retinopathy, neovascular glaucoma, rheumatoidarthritis, and psoriasis. The ability of EPO to reduce or preventabnormal angiogenesis will be of use in preventing or reducingangiogenesis associated with such disease states.

One method according to the present invention is the use of EPO as anadjunct in the chemotherapy of neoplastic disease. EPO is provided inendothelial-protecting amounts where protection of the endothelium fromthe adverse effects of chemotherapeutic agents is desired. A secondmethod according to the present invention is the use of EPO as anadjunct in the chemotherapy of neoplastic disease, where enhancement ofthe adverse effects of chemotherapeutic agents on endothelium (e.g.,enhancement of endothelial growth suppression) is desired. In suchsituations, EPO is provided in endothelial-inhibiting amounts.

As used herein, endothelial-protecting amounts of EPO refer to thosedosages which reduce or prevent the suppression of endothelial growthwhich would otherwise occur due to exposure to a chemotherapeutic agentor radiation, mechanical trauma, or a disease state known to damage theendothelium. Alternatively, an endothelial-protecting amount of EPO maybe defined as those dosages which increase the numbers of viableendothelial cells following exposure to the chemotherapeutic agent orradiation, mechanical trauma, or a disease state known to damage theendothelium; the increased number of viable cells is in comparison tothat which would be expected in the absence of EPO. The most effectiveendothelial-protecting amounts of EPO may vary depending upon the timeof administration and the etiology of endothelial damage.

Where endothelial damage is due to exposure to a chemotherapeutic agent,the most effective endothelial-protecting amounts of EPO will varydepending upon whether EPO is administered simultaneously with, priorto, or after, the chemotherapeutic agent, and may vary depending uponthe specific chemotherapeutic agent in question.

As used herein, endothelial-inhibiting amounts of EPO refer to thosedosages which enhance or increase the suppression of endothelial growthwhich would otherwise occur due to exposure to a chemotherapeutic agentor radiation, mechanical trauma, or a disease state known to damage theendothelium. Alternatively, an endothelial-inhibiting amount of EPO maybe defined as those dosages which decrease the numbers of viableendothelial cells following exposure to the chemotherapeutic agent orradiation, mechanical trauma, or a disease state known to damage theendothelium; the decreased number of viable cells is in comparison tothat which would be expected in the absence of EPO. The most effectiveendothelial-inhibiting amounts of EPO may vary depending upon the timeof administration and the etiology of endothelial damage.

Where endothelial damage is due to exposure to a chemotherapeutic agent,the most effective inhibiting amounts of EPO will vary depending uponwhether EPO is administered simultaneously with, prior to, or after, thechemotherapeutic agent, and may vary depending upon the specificchemotherapeutic agent in question.

Endothelial damage may be assessed by a reduction in the proliferationof endothelial cells and/or decreased numbers of viable endothelialcells, leading to a total decrease in the number of viable endothelialcells. Such a decrease in the number of viable endothelial cells mayalso be referred to as endothelial growth suppression, or endothelialcell suppression or inhibition.

As used herein, a method of reducing endothelial injury in a subjectcaused by administration of a chemotherapeutic agent to the subjectrefers to a method which reduces or prevents the decrease in viableendothelial cells which would otherwise be caused by administration ofthe chemotherapeutic agent. As used herein, a method of enhancingendothelial cell inhibition in a subject caused by administration of achemotherapeutic agent to the subject refers to a method which increasesor enhances the reduction in viable endothelial cells which wouldotherwise be caused by administration of the chemotherapeutic agent.

Damage to endothelial cells may also be caused by radiation therapy,mechanical trauma, and by disease states such as inflammation, heartdisease (e.g., atherosclerosis) and cancer. In atherosclerosis, forexample, injury to or dysfunction of the endothelium leads to reducedvasodilator response and to increased platelet deposition on thearterial wall. Serotonin and thromboxane A₂ released from depositedplatelets cause arterial constriction and spasm, increase adhesion andaggregation of platelets, and enhance the atherosclerotic process. Theconsequences of coronary obstruction are often ameliorated by theformation of new coronary vessels in response to angiogenic stimuli. Theuse of EPO to enhance endothelial growth and/or repair, or to preventendothelial damage, will be a useful adjunct in treating endothelialdamage due to mechanical damage, radiation therapy, or due to diseasestates which adversely affect the endothelium.

As used herein, human erythropoietin (EPO) refers to both the naturallyoccurring human erythropoietin glycoprotein as well as recombinant humanerythropoietin (rHuEpo or epoetin alfa, available commercially asEpogen® (Amgen Inc., Thousand Oaks, Calif.) and as Procrit® (OrthoBiotech Inc., Raritan, N.J.)). Peptide analogs of EPO may also be usedin the methods of the present invention. As used herein, peptide analogsare those compounds which, while not having amino acid sequencesidentical to that of EPO, have a similar three-dimensional structure. Inprotein molecules which interact with a receptor, the interaction takesplace at the surface-accessible sites in a stable three-dimensionalmolecule. By arranging the critical binding site residues in anappropriate conformation, peptides which mimic the essential surfacefeatures of EPO binding region may be designed and synthesized inaccordance with known techniques. A molecule which has a surface regionwith essentially the same molecular topology to the binding surface ofEPO will be able to mimic the interaction of EPO with the EPO receptor.Methods for determining peptide three-dimensional structure and analogsthereto are known, and are sometimes called ‘rational drug designtechniques’. See, e.g., U.S. Pat. No. 4,833,092 to Geysen; U.S. Pat. No.4,859,765 to Nestor; U.S. Pat. No. 4,853,871 to Pantoliano; U.S. Pat.No. 4,863,857 to Blalock (applicants specifically intend that thedisclosures of all U.S. patents cited herein be incorporated byreference in their entirety).

Peptides which mimic the biological activity of erythropoietin (EPOreceptor peptide ligands) may be substituted for EPO in the methods ofthe present invention. The sequence of such peptides may representfragments of the full-length EPO protein sequence, which fragments arecapable of binding to and activating the EPO receptor. Additionally,peptides with sequences dissimilar to that of EPO may be utilized in themethods of the present invention, where such peptides mimic thebiological activity of EPO. Wrighton et al. report the identificationand characterization of small peptides that bind to and activate theerythropoietin receptor on the surface of target cells, although thepeptides' sequences are not similar to the primary sequence of EPO(Wrighton et al., Science 273:458 (26 Jul. 1996)). These peptideagonists are represented by a 14-amino acid disulfide-bonded cyclicpeptide with an identified minimum consensus sequence. The structure ofa complex of one such peptide mimetic with the erythropoietin receptoris described by Livnah et al., Science 273:464 (26 Jul. 1996).

As used herein, the term chemotherapeutic agent refers to cytotoxicantineoplastic agents, that is, chemical agents which preferentiallykill neoplastic cells or disrupt the cell cycle of rapidly proliferatingcells, used therapeutically to prevent or reduce the growth ofneoplastic cells. Chemotherapeutic agents are also known asantineoplastic drugs or cytotoxic agents, and are well known in the art.As used herein, chemotherapy includes treatment with a singlechemotherapeutic agent or with a combination of agents. In a subject inneed of treatment, chemotherapy may be combined with surgical treatmentor radiation therapy, or with other antineoplastic treatment modalities.

Exemplary chemotherapeutic agents are vinca alkaloids,epipodophyllotoxins, anthracycline antibiotics, actinomycin D,plicamycin, puromycin, gramicidin D, paclitaxel (Taxol®, Bristol MyersSquibb), colchicine, cytochalasin B, emetine, maytansine, and amsacrine(or “mAMSA”). The vinca alkaloid class is described in Goodman andGilman's The Pharmacological Basis of Therapeutics, 1277-1280 (7th ed.1985) (hereafter “Goodman and Gilman”). Exemplary of vinca alkaloids arevincristine, vinblastine, and vindesine. The epipodophyllotoxin class isdescribed in Goodman and Gilman, supra at 1280-1281. Exemplary ofepipodophyllotoxins are etoposide, etoposide orthoquinone, andteniposide. The anthracycline antibiotic class is described in Goodmanand Gilman, supra at 1283-1285. Exemplary of anthracycline antibioticsare daunorubicin, doxorubicin, mitoxantraone, and bisanthrene.Actinomycin D, also called Dactinomycin, is described in Goodman andGilman, supra at 1281-1283. Plicamycin, also called mithramycin, isdescribed in Goodman and Gilman, supra at 1287-1288. Additionalchemotherapeutic agents include cisplatin (Platinol®, Bristol MyersSquibb); carboplatin (Paraplatin®, Bristol Myers Squibb); mitomycin(Mutamycin®, Bristol Myers Squibb); altretamine (Hexalen®, U.S.Bioscience, Inc.); cyclophosphamide (Cytoxan®, Bristol Myers Squibb);lomustine [CCNU] (CeeNU®, Bristol Myers Squibb); carmustine [BCNU](BiCNU®, Bristol Myers Squibb).

Methods of administering chemotherapeutic drugs vary depending upon thespecific agent used, as would be known to one skilled in the art.Depending upon the agent used, chemotherapeutic agents may beadministered, for example, by injection (intravenously, intramuscularly,intraperitoneally, subcutaneously, intratumor, intrapleural) or orally.

As used herein, the administration of a compound “in conjunction with” asecond compound means that the two compounds are administered closelyenough in time that the presence of one alters the biological effects ofthe other. The two compounds may be administered simultaneously(concurrently) or sequentially. Simultaneous administration may becarried out by mixing the compounds prior to administration, or byadministering the compounds at the same point in time but at differentanatomic sites or using different routes of administration.

The phrases “concurrent administration”, “simultaneous administration”or “administered simultaneously” as used herein, means that thecompounds are administered at the same point in time or immediatelyfollowing one another. In the latter case, the two compounds areadministered at times sufficiently close that the results observed areindistinguishable from those achieved when the compounds areadministered at the same point in time.

Subjects to be treated by the method of the present invention includeboth human and animal (e.g., dog, cat, cow, horse) subjects, and arepreferably mammalian subjects.

Many chemotherapeutic agents act at specific phases of the cell cycle,and are active only against cells in the process of division. Neoplasmswhich are the most susceptible to chemotherapy are those with a highpercentage of cells in the process of division, including but notlimited to breast, liver, brain, lung, and ovarian cancer. Highlyvascularized solid tumors are amenable to treatment withendothelial-inhibiting amounts of EPO in conjunction withchemotherapeutic agents, as these tumors rely on angiogenesis to provideadequate blood supply to the growing tumor tissue.

EPO used according to the methods of the present invention may beadministered by any suitable means, as would be apparent to one skilledin the art. EPO may be administered systemically (e.g., intravenously)or locally (e.g., injected into a tumor, tissues immediately surroundinga tumor, or into an anatomic compartment containing a tumor). Forexample, where an endothelial-inhibiting amount of EPO is utilized as anadjunct to chemotherapy, the EPO may be administered locally to a tumor(or the immediately surrounding tissue) in which it is desirable toprevent angiogenesis. Where a chemotherapeutic agent is deliveredsystemically, for example, an endothelial-protecting amount of EPO maybe administered systemically by intravenous injection.

The dosage and timing of EPO administration used in conjunction with achemotherapeutic agent will similarly depend upon the desired effect.The present inventors have discovered that depending upon the timing ofEPO administration (simultaneous with, before, or after chemotherapeuticagent administration) and the dosage of EPO, EPO either protects theendothelium from the growth-inhibiting effects of chemotherapeuticagents, or enhances the endothelial growth inhibition seen withchemotherapeutic agents. It will be apparent to those skilled in the arthow to determine, by routine experimentation, the dosage and timing ofEPO administration in conjunction with a particular chemotherapeuticagent to achieve a desired effect.

The maximum amount of EPO that can be administered in single or multipledoses has not been determined. Doses of up to 1,500 Units/kg for threeto four weeks have been administered without toxic effects due to EPOitself. Eschbach et al., in: Prevention of Chronic Uremia (Friedman etal., eds.), Field and Wood Inc., Philadelphia, pp. 148-155 (1989). Inthe present methods, where it is desired to protect the endothelium fromthe endothelial damage and/or endothelial growth suppression caused by achemotherapeutic agent, EPO is administered in an endothelial-protectingamount. Suitable endothelial-protecting dosages may range from about 100U/kg to about 200 U/kg. In the present methods, where it is desired toenhance the endothelial damage and/or endothelial growth suppressioncaused by a chemotherapeutic agent, EPO is administered in anendothelial-inhibiting amount which may range from about 750 U/kg toabout 2,000 U/kg. As noted above, the dosage and timing of EPOadministration used in conjunction with a chemotherapeutic agent willdepend upon the desired effect, as well as the chemotherapeutic agentutilized.

The following examples are provided to illustrate the present invention,and should not be construed as limiting thereof.

Example 1 Materials and Methods

Cell Culture. Human umbilical vein endothelial cells (HUVECs) wereobtained from Caesarian section derived cords. HUVECs were cultured bystandard methodology in 25 cm² T-flasks (Corning Inc., Corning, N.Y.)coated with 0.5% porcine skin gelatin (Sigma Chemical Co., St. Louis,Mo.). Medium 199 (Life Technologies, Gaithersburg, Md.), supplementedwith 20% defined fetal bovine serum (FBS) (Hyclone, Logan, Utah), 16U/ml heparin (Sigma), 50 μg/ml bovine hypothalamus derived endothelialmitogen (Biomedical Technologies, Stoughton, Mass.), 100 U/ml penicillinand 100 μg/ml streptomycin was used for the growth of HUVECs.Endothelial cells were characterized by homogenous and typicalcobblestone morphology, von Willebrand factor antigen positivity, andthe presence of Weibel-Palade bodies, as are known in the art.

Protection/Inhibition Assay. The number of metabolically active cellsafter exposure of endothelial cell cultures to test agents was assessedusing a calorimetric method. This assay utilizes solutions of atetrazolium compound[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4Sulfophenyl)-2H-tetrazolium] (MTS) and an electron coupling reagent,phenazine methosulfate (PMS; available from Promega Corp., Madison,Wis.). See Denizot and Lang, J. Immunol. Methods 89:271 (1986); PromegaCorporation Technical Bulletins 112, 152 and 169). MTS is bioreducedinto a formazan by dehydrogenase enzymes found in metabolically activecells. The amount of formazan is measured at 490 nm absorbance and isdirectly proportional to the numbers of living cells in culture.

Endothelial cells grown in the complete (supplemented) M199 medium wereharvested in the log phase. At 80-90% confluency, EC culture monolayerswere washed with phosphate buffered saline (PBS), treated with 0.25%trypsin in 1 mM EDTA for 1-2 minutes, and then the cells were suspendedin complete medium The number and viability of the cells was determinedusing a hemocytometer and the trypan blue staining, respectively. Cellsuspensions of 7.22×10⁴ cells/ml medium were prepared and 90 μl (6.5×10³cells) were dispensed into each well of a 96-well plate. After overnightincubation at 37° C., 5% CO₂, in a humidified atmosphere, EPO and/or thechemotherapeutic agent were added at concentrations and in the orderspecified in the examples described below. Plates were then incubatedfor another 24 hours. At the end of this incubation period, 20 μl offreshly prepared combined MTS/PMS (20:1 ratio) solution was added intoeach well and the plates were incubated for 1-4 more hours, as permanufacturer's recommendations. The absorbance of each well at 490 nmwas recorded using an ELISA plate reader. The LD50 and the effect of thevarious treatments on cell viability and chemosensitivity weredetermined by plotting the corrected absorbance at 490 nm versus theconcentration of the additive (EPO, chemotherapeutic agent, orcombinations thereof).

Statistical Considerations. For protection/inhibition assays,experiments were performed in triplicate. All other experiments wereperformed at least five times. Results were averaged and means±SDreported. Controls for all experiments included one to two triplicatewells treated with each of the following:

1) 1 μg/ml cisplatin;

2) 50 μg/ml cisplatin;

3) 10 or 20 U/ml of EPO;

4) 0.6 or 1.2 U/ml of EPO.

Thus for each experiment, three to six wells received the above fourcontrol treatments (total 12-24 control wells). An additional controlconsisting of a triplicate well of untreated cells was also performed.

Example 2 Determination of Cisplatin LD50

Ninety-six-well plates containing endothelial cells were prepared asdescribed in Example 1 and incubated overnight at 37° C., 5% CO₂, in ahumidified atmosphere. A solution of 160 μg/ml cisplatin was prepared,and serial dilutions were added to the wells (5 μl per well;concentrations varied from 0.03125 μg/ml to 4.0 μg/ml. The plates werethen incubated for two days (48 hours) and viability of endothelialcells was assessed using the MTS/PMS technique described in Example 1.The absorbance of each well at 490 nm was recorded using an ELISA platereader. The corrected absorbance at 490 nm versus the concentration ofcisplatin (μg/mL) was plotted (FIG. 1) to provide a dose-response curve.The concentration of cisplatin required to give 50% of the maximalresponse (LD50 of cisplatin) was determined to be 0.45 μg/ml.

In view of the above findings, a dosage of 1 μg/ml of cisplatin was usedto determine the effects of EPO on endothelial cells, as provided in thefollowing examples.

Example 3 Effects of Simultaneous Cisplatin and EPO on Endothelial Cells

To determine the effects of combined EPO and cisplatin on endothelialcells, serial dilutions of EPO were added to endothelial cell culturessimultaneously with cisplatin.

Endothelial cell cultures were prepared as described in Example 1.Cisplatin (final concentration of 1 μg/ml) was added to each test wellsimultaneously with 5 μl of various EPO preparations (final EPOconcentration ranging from 0.15 to 20 U/ml. Endothelial cell viabilitywas assessed using the MTS/PMS calorimetric assay described inExample 1. Results were compared to control wells (endothelial cellstreated with 1 μg/ml cisplatin alone, considered as the baseline andrepresented in FIG. 2 as 0%). Results are provided in FIG. 2; the “% ofcontrol” is the percentage change of optical density at 490 nm over thecontrol, such that “0%” indicates the test well had similar numbers ofmetabolically active cells as the control, whereas “50%” indicates 50%more and “−50%” indicates 50% fewer metabolically active cells.

As shown in FIG. 2, a biphasic response was observed when EPO was addedto cell cultures simultaneously with the addition of cisplatin.Endothelial cell cultures treated with from 0.15 to 1.25 U/ml of EPOwere protected from the damaging effects of cisplatin when EPO was addedsimultaneously with cisplatin. EPO concentrations of 0.3 U/ml providedthe greatest protection of endothelial cells when EPO was addedsimultaneously with cisplatin; the number of viable cells wasapproximately 30% greater than that observed in control cultures treatedwith cisplatin only.

As also shown in FIG. 2, endothelial cell growth was inhibited incultures treated with from 5 to 20 U/ml of EPO when EPO was addedsimultaneously with cisplatin, compared to cultures treated withcisplatin alone. Cultures treated with 5 U/ml of EPO and 1 μg/mlcisplatin showed a 33% decrease in the number of viable cells comparedto control cells exposed to cisplatin alone.

Example 4 Effects of EPO on Endothelial Cells Administered afterCisplatin Exposure

In this experiment, serial dilutions of EPO were added to endothelialcell cultures two hours after the cultures were exposed to cisplatin.

Endothelial cell cultures were prepared as described in Example 1.Cisplatin was added to each test well (1 μg/ml final concentration ofcisplatin); two hours later 5 μl of an EPO preparation ranging from 0.15to 20 U/ml final concentration was added. Endothelial cell viability wasassessed using the MTS/PMS calorimetric assay described in Example 1.Results were compared to control wells (endothelial cells treated with 1μg/ml cisplatin alone).

Results are provided in FIG. 3, and show that a biphasic response wasobserved when EPO was added to cell cultures after the addition ofcisplatin. Endothelial cell cultures treated with from 0.15 to 5 U/ml ofEPO were protected from the damaging effects of cisplatin when EPO wasadded two hours following cisplatin exposure. The number of viable cellsafter treatment with 1.25 U/ml EPO after cisplatin exposure was 34%greater than that of controls. In contrast, cell viability in thepresence of 10 to 20 U/ml EPO administered two hours after cisplatinexposure was reduced over that seen in controls (cisplatin only).

Example 5 Effects of EPO on Endothelial Cells Administered Prior toCisplatin Exposure

In this experiment, serial dilutions of EPO were added to endothelialcell cultures two hours before the cultures were exposed to cisplatin.

Endothelial cell cultures were prepared as described in Example 1. Eachtest well received 5 μl of an EPO preparation ranging from 0.15 to 20U/ml EPO; two hours later cisplatin was added to each test well (5 μl of1 μg/ml cisplatin). Endothelial cell viability was assessed using theMTS/PMS calorimetric assay described in Example 1. Results were comparedto control wells (endothelial cells treated with 1 μg/ml cisplatinalone).

Results are provided in FIG. 4, and show a reduction in the number ofviable endothelial cells after exposure to EPO two hours prior tocisplatin exposure (compared to control cells exposed only to cisplatin)Cell proliferation and viability was decreased by as much as 81%compared to controls. The inhibition was dose dependent; EPOconcentrations as low as 5 and 2.5 U/ml reduced cell growth by 58% and48%, respectively, compared to controls.

The foregoing is illustrative of the present invention and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1-15. (canceled)
 16. A method of treating endothelial injury in asubject, comprising administering an effective endothelial-protectingamount of erythropoietin to said subject in need of such treatment,wherein said endothelial injury is caused by mechanical damage, exposureto radiation, exposure to chemotherapeutic agents, inflammation, heartdisease or cancer.
 17. The method according to claim 16, wherein saidendothelial injury is caused by mechanical damage.
 18. The methodaccording to claim 16, wherein said endothelial injury is caused byexposure to radiation.
 19. The method according to claim 16, whereinsaid endothelial injury is caused by inflammation.
 20. The methodaccording to claim 16, wherein said endothelial injury is caused byheart disease.
 21. The method according to claim 16, wherein saidendothelial injury is caused by cancer.
 22. The method according toclaim 16, wherein said erythropoietin is administered intravenously orsubcutaneously.
 23. A method of treating endothelial injury in asubject, comprising administering an effective endothelial-protectingamount of erythropoietin to said subject in need of such treatment,wherein said effective endothelial-protecting amount of erythropoietinreduces or prevents the suppression of endothelial growth associatedwith endothelial injury caused by mechanical damage, exposure toradiation, exposure to chemotherapeutic agents, inflammation, heartdisease or cancer.
 24. A method of treating endothelial injury in asubject, comprising administering an effective endothelial-protectingamount of erythropoietin to said subject in need of such treatment,wherein said effective endothelial-protecting amount of erythropoietinincreases the numbers of viable endothelial cells following endothelialinjury caused by mechanical damage, exposure to radiation, exposure tochemotherapeutic agents, inflammation, heart disease or cancer.
 25. Themethod according to claim 16, wherein said erythropoietin isadministered in an amount of from about 100 Units per kilogram to about200 Units per kilogram.
 26. A method of reducing endothelial injury in asubject caused by administration of cisplatin to the subject, comprisingadministering an effective endothelial-protecting amount oferythropoietin in conjunction with the administration of cisplatin. 27.The method according to claim 26, wherein said erythropoietin isadministered intravenously or subcutaneously.
 28. The method accordingto claim 26, wherein said effective endothelial-protecting amount oferythropoietin is administered simultaneously with said cisplatin. 29.The method according to claim 26, wherein said effectiveendothelial-protecting amount of erythropoietin is administered prior tosaid cisplatin.
 30. The method according to claim 26, wherein saideffective endothelial-protecting amount of erythropoietin isadministered after said cisplatin.